J. Mol.
Biol.
(1976)
101, 503-536
Developmental Biochemistry of Cotton Seed Embryogenesis and Germination VI1.t Characterization
of the Cotton Genome
VIRGINIA WALBOT~ AND L. S. UURE III$ I)epartment
of Biochemistry.
(Received 30 April
Univer&y
nf (r’eorqia, Athew,
(!a 30602, [J.S.A.
197.5, and in revised form 20 September 1975)
The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, baxe composition, and genetic complexity. The haploid genome size is found to be 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due t,o trrrdored~lplicatic)n of the entire penomo. Non-repetitive deoxynucleotide sequences account for approximately 60.5qO of the cotton genome (C& pure 7 = 437); highly repetitive sequences ( > 10,000 r(bpetition frequency) constitute about 7.7 Y. of tile penomc (C& pure = 4.6X IO 4, and intermediately repetitive sequences constitute tile remaining 27 “/b of cytoplasmic ribottle genome (C,t+ pure = 1.46). Hybridizat,ion of ‘251-labrletl somal RNA to whole-cell DNA on filters and in solution indiratr approximately 300 to 350 copies of the rRNA cistrons per haploid genome. The interspersion of repetitive sequences that reassociate between C,t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined t)y determining t,he reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the penomr consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 200/b of the genome may bc involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence di\.cLrpence is evident, among t,he members of this component. Highly repetitive sequence elements that are reassociated by a C,t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of tllis highly repetitive component.
11Abbreviations used: C,t, product of DNA concentration (mol nucleotido I ‘) x time(s); C,t,, that C,t value at which one-half of a kinetic component of DNA will have reassociated: Coti pure, that C,t value at which one-half of a kinetic component of DNA would have re: associated if only thet component were present, calculated as the product of Cot l x the fraction of t’he genome in that kinetic component; 5 MeC, 5.met,hylcytosine; rDNA, ribosomal RNA cist,rons. 503
1. Introduction All the genomes of euknryotes thus far examined contain both repetit#ive and nonrepetitive sequence elements. The non-repetitive sequence elements are thought to include t’he structural genes. There is some evidence for t,his hypothesis, since the messenger RNAs coding for specific proteins are transcribed from non-repetitive DNA (globin, Bishop & Freeman, 1973: Bishop & Rosbash. 1973; Harrison af al.. 1974: ovalbumin, Harris ef al., 1973: fibroin. Suzuki et aZ., 1972). Furthermore, mRNA isolated from various tissues has been shown t,o be transcribed primarily from non-repetitive DNA sequences, although some repetitive copy transcript is also present (Greenberg & Perry, 1971; Goldberg et al., 1973; Firtel & Lodish, 1973: Bishop et al., 1974: Campo & Bishop. 1974; Galau et al., 1974: Klein et al.; 1974: Spradling et al., 1974; Davidson et al.. 1975). Messenger RNA for histone proteins has been shown to be transcribed from repetitive copy DNA (Weinberg et al., 1972). The proportion of transcripts of repetitive DNA elements in heterogeneous nuclear RNA is higher than from mRNA from t’he same tissue (,Jelinek et ~2.. 1973; Holmes & Bonner, 1974; Smith et al., 1974) suggesting that initial transcriptional products contain more RNA transcribed from repetitive DNA than does functional mRNA. The two kinetic components of the eukaryoOic genome are interspersed to some extent in all the animal genomes which have been analyzed in d&ail. Interspersion has been demonstrated (1) by the increasing proportion of DNA binding to hydroxylapatite at low C,t values? with DNA fragments of increasing length (calf, Britten & Smith, 1970: Xenopus, Davidson et al., 1973: sea urchin, Graham et al.. 1974: Oncopelfus fusciatus, Lagowski et al., 1973: chicken, de *Jimenez et al.: 1974), (2) by electron microscopy of reassociated DNA (rat liver, Bonner sf al., 1973: DrosophiZa, Manning et al.? 1975) and (3) by inference from the presence of both repetitive and non-repet’it,ive sequences transcribed into the same RNA molecules (Dictyostelium, Firtel & Lodish, 1973). In both Xenopus (Davidson et al.: 1973) and sea urchin (Graham et al.. 1974) short repetitive sequences of about 300 base-pairs are interspersed with longer non-repetitive sequences of about 1000 base-pairs in over 5Oy{, of the genome. Regions of clustered repetitive sequences, a longer interspersion pattern (repetitive DNA elements more than 4000 bases apart) and long sttret8ches of non-repet’itive DNA also probably exist. .It is attractive to view t’he interspersion pattern of repetitive a,nd non-repetitive sequence elements as a contiguous arrangement of regulatory (repetitive) and structural gene (non-repet,itive) sequences (reviewed by Davidson & Britten, 1973). This notion is strengthened by the fact that most mRNA sequences in SW urchin gastrulae are transcribed from non-repctitivct DNA adjaccant, to repetitIivcb DNA (Davidson et wZ., 1975). t Abbreviations used: C,t, product of Di’GA concentration (mol nucleotlde 1 -‘) x time(s); C,t*, that Cot value at which one-half of a kinetic component of DNA will have reassociated; Cat+ pure, that Got value at which one-half of a kinetic component of DNB would have reassociated if only that component were present,, calculated as the product, of C,l& x the fraction of the genome in that kinetic component: 5 MeC, B-methylcytosine; rDNA, ribosomal RNA oistrons.
(‘HARBC’TERIZSTIOK
OY THE
GROTTOS
GENOME:
50.5
Although the presence of repetitive and non-repetitive DI\;A has also been report.cd in higher plants (Britten & Kohne, 1967; Sivolap & Bonner, 1971; Millerd & Whitfeld. 1973; Flavell et al., 1974; Cullis & Schweizer, 1974; Nze-Ekekang et al., 1974; Ranjekar ut al.. 1974) bht: possible interspersion of t,hese components has not been studied. Tn this paper we present evidence demonstrating the ext,ensive int’erspersion of thcb mildly,, repctit,irfB and non-rep&t ive sequcnc(’ elements of t,he cot’ton penomr:.
2. Materials and Methods (a) Plant material (:rt,c~rlliouse-g~,(~~~~ plants of ~hssypium I&SU~WL ((‘oker var. %Ol) provided a contillnous suppl?- of developing embryos md mature seed. The growt’il rate of cott,on embryos insidtx the cott,on boll is shown in Fig. 1 ; sevc~ral hiochemion1 paralncxtcrs of elnbryo dc\-elopmont Iravc IHWI studied (Dure. 1973). (I)) Cell num6er
tleter),li)lntiorrs
Cotyledons from embryos dissected at various stages of development were digest,rd for 48 h in 596 cllromic acid to disrupt the t,issue t,o sirlgle cells (Brown & Rickless, 1949). The cotyledons consist of epidermal, parenchymal and vascular cells. Dilutions of the ccl1 suspensions were counted in a Fuchs-~Rojenthal hemacytometer at a magnification of 100. From 5 to 10 dilutions were made of each cotyledon cell suspension and 5 port’ions of eaclr dilution were countsed. Approximately 2:; of the cells are vascular cells, xylem oleme,lts and sieve tubes, t-hat do not contain an)- cell orga,nelles or nucleic acids; thesr cells \\.erc tlot incaludcd in the total cell number. since tllsy do not) contribute to total DNA c!clut~nlll.
(c) D,VA
extraction.
purijcation
and~rag,nentation
DNA was prepared from embryonic cotyledons at various stages of development. from cotyledons of the mature seed. and from germinating root tips. Homogenizat’ion was carried out in 1 x SSC (SSC is 0.15 M-NaCl, 0.015 Al-trisodium citrate), 2.57; sodium dodecyl sulfate, 1 mM-EDTA (25 g fresh wt/250 ml) with a Polytron homogenizer (Brinkman Instruments) at top speed for 2 min. The homogenate was stirred at room temperaturtj for several hours. then made 1 31 in NaC104 and heated to 60°C for 15 min. Two vol. chloroform/inoamyl alcohol (24:l) were added to the Ilomogenate and the mixture stirred for sex-era1 hours. The aqueous phase was recovered b>- centrifugation at 5000 g for 10 milt and re-extracted wit,h chloroform/isoamyl alcohol for I h. The aqueous phase was again recovered as described above and the nucleic acids precipitated by the addition of 2.5 vol. cold ethenol. The precipitate was resuspended in 20 ml of 1 >; SW and incubated with 2 mg pancreatic RNcse (Sigma), boiled for IO min prior to use. and 400 units T, RNastb (Wort~llingt~on) for 2 11 at 37”(.‘. Aft)er several additional chloroform/isoamyl alcohol extra&ions and ethanol precipitations, from which tllr: DNA was collected by spooling, t,he DNA was purified from residual oligoribonucleotides by pmcipitat,ion with 0.54 vol. isopropanol (Marmur, 1961). Finally the DNA was banded in preparative (IsCl gradients h5, centrifugation at 75,000 g for 60 h at 22°C. Gradient fractions containing DNA were pooled. dialyzed extensively against 1 x SSC, and stored as &anol precipitates. The, weight average molecular weight of native DNA samples was determined b!, sedimentation analysis with a Spinco model E analytical ultracent~rifuge equipped wit11 an ultra.violet light scanner, using the equations given by Studier (1965). Single-stranded weight average molecular weight was determined in O.!) .w-NaCI, 0.1 &I-NaOH, and doublest,randed molecular weight in 1-O M-NaCl. Three determinations were made for each sample. The weight average molecular weight of native DNA isolated b>- our procedure nvcragtxd 1 > 107. Singl+stranded molecular wcaiphts averaged al)ont 40 to X0, of tllr nativcl DNA \‘HIll(~S. :i I
306
\‘.
\\‘;\l,BOT
AXI)
L.
8. DURE
III
DNA blloyatlt tlensit,y \WS tlrtrrmined by isopycnic C&l centrifugation by the method of Szybalski & Szybalski ( 1971) using a Spinco model E ultracentrifuge equipped with a DNA was used as a marker (1.731 g cmW3); data reported u.\-. scanner. ~~icrococcus l&us are the average of 3 or more crntrifugations on 2 or more separately prepared DNA samples. The optical charact,eristics of cotton DNA were obtained with a Gary 15 spectrophotoInetcr. The percentage hyprrchromicity and melting temperature (t,) were determined by melting DNA samples in a water-,jackcttcd cuvette attached to a Lauda K2jR circnlnting water bath. A thermistor probe (YSI model 402) was inserted into the solution tin-ough a Teflon plug at, tllc? top of thr cuvet~te. The probta was connected to a telethermonlrtrr (YSl model 4286) and temperature readings recorded manually. The dispersiotl, (T2,3r was determined as described 1)~ Mahlrr & D~ltton (1964). Escherichin coli DNA bvas optically analyzed as a standard. Native DNA was sheared to various sizes by sonic&ion, by passage tllrough a 25 gauge syringa needle, or by higll-speed blending in a Virtis llomogenizer as described by Britten et a/. (1974). Ttrr single-stranded lrr@lr of t,he DNA fragments was dct~erminrd in isoet ul.. 197X) 01' kinetic SIIC~OSO gradients corltainillg 0.1 Izr-NaOH (Noll, 1967 ; Davidson I)y analyt,ical ~iltrac~rltrif~l~at,ioll. The equations relating molt~cutar wtaight to srdimerltation rate (Studier, 1965) \vcArcaderived for molecules more than an order of mapnitllde larger than the smallest molecules used in t’his st,udy ; thus. the measurements reported reflect relative size relationships, rather t,han ttle absolute molecular size. Fragments produced by all 3 methods of shearing showed thermal denaturatioll charactorist,ics similar to nativrl cotton DNA (Fig. 3(a)), indicating t)bat the> fragmentas were ttssontiatly undenatured. (d) lo&nation
of nucleic
ucirl~s
Heat,-denatured DNA alld purified rRNA were iodinated by tlje procedure of C&z et ul. lzsI ( 17.7 Ci/mmol, New England Nuclear) itI ( l!j72) wit11 the following modifications. NaOH was neut,ralized wit11 0.1 M-HCl in 0.005 M-Na,SO, to reduce the-ioditie to bypoiodate, the reactive oxidation stat,e for t,he iodination reaction; the thallimn chloride (K & K Laboratories) concentration was reduced to 10m4 ~1. Following the reaction, the iodinatc>d nucleic acids were separated from other reaction components by chromatography on Hephadex G25 in 0.1 x SSC and their molecular weight determined again. Short fragment length DNA ( < 1600 nucleotidos) showed no detectable reduction in size during iodination ; however, longer fragment length DNA did (30% reduction at initial length of 9200 nuclootides). The ribosomal RNAs showed some decrease in molecular weight after iodination, as shown hy their rnigratioll on polyacrylamide gels. DNA iodinatod in this way did not exhibit any change in specific activity ( + 2yo) after boiling for 10 min in a sealed capillary or prolonged incubation at 60°C in 0.12 >l-PB buffer (equimolar amount,s of mono- and dibasic phosphate buffer, pH 6.8). Cotton DNA is approximately 130/b cytidine and 4.6% 5.methylcytidine (see Results). Assuming that the 5-MeC residues are not iodinated, a spec. act. of 5 x lo5 cts/min per pg DNA is equivalent to one [1251]iodocytidine and 21 iodocytidine residues for every 3800 cytidine residues. Iodination is assumed to be basically a random process, at least with respect, to the labeling of the major kinetic components of cotton DNA. This was confirmed by the fact that the specific activities of mildly repetitive sequence DNA (reassociating between C,t values of 0.1 and 50) and non-repetitive sequence DNA (not reassociated by Cot z 50) were identical. However, since the iodination react’ion entails an incubation that equates to a C,t value of 0.05, the most rapidly reassociating DNA component (zero-time: binding which, in turn, could lead to an DNA) may not participate in the iodination reaction, underestimation of the magnitude of this component when measured isotopically.
(e) DNA/DNA
reaesociation
The rate of reassociation of heat-denatured DNA fragmented to several size classes was determined. Portions of sheared DNA were sealed in sterile capillary tubes or in ampules, denatured in a boiling water bath for 7 min, and then allowed to renature for
(‘HARACTERIZATION
OF
THE
COTTON
GENOME
507
varying lengths of time. The reassociation reactions were terminated by freezing the samples at - 20°C. The criterion conditions (Britten & Kohne, 1967) for these experiment,s were 0.12 M-PB buffer, 60°C (t,-225 deg. C). Various concentrations of SSC and PB buffer were used in the renaturation experiments and all values reported have been corrected to the Cot value equivalent to criterion conditions. In addition, the apparent rate of reassociation has been corrected to accolmt for the low G $ (’ content of cott,on DNA using the formula of Wetmur & Davidson (1968). Reassociated molecules were separated from single-stranded molecules on hydroxylapatite (BioRad) columns prepared in 0.12 M-PB buffer and 0.1% sodium dodecyl sulfat)t! at 60°C’. The hydroxylapatite was boiled for 5 min prior to pouring into the column and the column was washed with 0.12 M-PB buffer to remove sodium dodecyl sulfate prior to loading the sample. Single-stranded molecules were eluted with 0.12 M-PB and thcl reassociated duplexes &ted with 0.48 M-PB (Britt’en et al., 1974). The DNA content of tlach fraction was determined spectrally and/or by the determination of lz51 cont,ent. Recovery of radioactivity and absorbancy units was 1OO& lOq/,. In experiments in which lz61-labeled DNA was used as tracer, it was less than O.OSqh the concentration of thaw unlabeled fragments. DNA of fragment length f-PB buffer and then the fraction elutinp in 0.48 M-PB buffer (2) collected and its radioactivity determined. In subsequent experiments, to rstimate the binding due to reassociation exclusive of Z, its contribution to the observed reassociation was stibtracted as described by Davidson et al. (1973). The proportion of DNA retained by the hydroxylapatite colllmn due to %: varies with fragment length (Fig. 4), and for experiments with larger fragment lengths (> 1500 nucleotides) zero-time binding DNA was removed from the lZ51-labeled preparations by recovering and losing the fraction not bound to the hydroxylapatite column at Cot < 10m5. These t This program was the “General Multiparametric Curve-Fitting Program CFTB” developed by L. M&es and published by the Computing Laboratory, Department, of Chemistry. Clarkson College of Technology, Potsdam, N.T. 13676, U.S.A.
5OS “&ripped”
1.. \V.AJ,HO’l preparations for wliich
((I.1 t,o W25yA)
(g)
.\SIl
1,. R. 1)UHE
contained a small \&able fraction no corrnct,iori Ilas t,etsri ma&~.
Characterization
oj” reasmciated
DNA
IL1 of zero-t*ime
hy S, m&ease
binding
material
digestion
DNA samples (0.5 to 1 mg DNA) reassociated to a Cot value of 0.1, 10 or 50 in 0.3 btNaCl, 0.01 M-PIPES buffer (Sigma), pH 6.7 (Davidson et al., 1973) were incubated uitli 200,000 units of S1 nuclcase (Miles Laboratories) in a solution containing 0.2 iw-NaCl, 0.0167 ~I-PIPES buffer, 0.067 mai-zinc acetate, aiid 1.85 mM-mercaptoethanol for 3 Ii at. 37°C’ in a scaled ampule. Tlrc digested samplos were loaded directly on a Sephadex G75 column equilibrated in 0.12 N- PB buffer. The void voliuno contained 8, nuclease-resistant duplex DNA with few, if any, single-stranded regions as determined by electron microscopy. The proportion of Xi nuclease-resistant to S,-sensititre material was determined by a comparison of the absorbance at 260 nm of the fractions containing duplex DNA to the totally included fraction whicli contained all the digestion products. In some experiments 1251-labeled DNA samples were used and the separation and the relative amounts of material contained in t’he 2 fract)ions determined hp monitoring radioactivit>y. (11) Electron
w~icroscopy
was performed as described 1,s Visualization of DNA samples by electron microscopy Davis et al. (1971). Aqueous samples containing 0.5 pg duplex DNA/ml, 0.5 M-ammonium acet,ate, 0.001 M-EDTA(Na,), 6 mg cyt~ochromo c/ml (Calbiochem), pH adjusted to 7.6, wt~~: spread on a 0.25 M-ammonium acet,ate Iiypophase. Samples containing both single and double-stranded DNA were prepared in a solution containing 0.5 pg DNA/ml, 0.1 M-Tris, 0.01 M-EDTA(Na,), 6 mg cytochrome c/ml and 32.5 46 formamide (pH 8.5). This COIICCILtration of formamide at room temperature mimics t,ticl criterion temperature for hydroxylapatite chromatography, since cacti percent by volume of formamidu reduces the t, by 0.72 deg. C (McConaughy et al., 1969). At the ionic strength of the samples, the t, of cotton DNA would be about 70°C. Ttlc: 3~2.5~4 formamido would reduce this to ahod 25 deg. C’. Thus mismatched duplexes 47”C, making room temperature roughly equal t,o t, kvould be maintained to t,ho same extent as under criterion conditions. These samples were spread over a hypopha,se containing 0.01 31.Tris, 0.001 nr-sodium acetate and Ifi”/; acotato and rotary formamide (pH 8.5). All samples wer(’ staiued wit,h 10V6 w-many1 shadowed with PtjPd (4: 1) prior to rxaminat,ion in a Philips 200 electron microscope. Lengths of cotton DNA molecules were calculated from calibrated diffraction gratings, and by comparison to +X 174 single-stranded, assuming 2.07 x 106 daltons of DNA/pin. closed circiilar DNA. 1.7 x 1O6 dalmns. or co1 El plasmid, dont)l~?-strRlldod closed circular DNA, 4.2 x IO6 daltorw. Tlic> two mrtliotls of cst,imating DNA lengtti agreed lvittiirr 5’j,,. (i) lZiboaoma1 RNA
%solation
Monomeric 80 S ribosomes were isolated from 3 g of cotyledons by liomogenizatiou in 24 ml of 0.1 nr-Tris (pH 8.0), 0.005 1~-M&l,, 0.01 M-KC1 and 0.5% Triton X-100 (Rohm & Haas), followed by layering 4-ml portions of t,ht: liomogenate on 3(1-d linear sucrose gradients (10% to 40% sucrose containiug 0.01 nr-Tris (pH 8.0), 0.005 %I-MgCl,, 0.01 M-KC]) and centrifugation for 6 h at 100,000 g in the Beckman SW27 rotor. Fractionation of the gradients was monitored at 260 nm and fractions containing 80 S ribosomes were pooled and the ribosomes pelleted by centrifugation at 250,000 g for 12 h at 4”C, in the Beckman Spinco 60Ti rotor. The ribosomal pellets were dispersed in 0.01 ivr-Tris (pH 7.6), 0.005 MMgCl,, 0.1% sodium dodecyl sulfate and extracted with buffer-saturated phenol for 30 min at 4°C. The aqueous phase was recovered by oentrifugation at 10,000 g for 10 min and the extraction repeated. The rRNA was precipitated with 2 vol. ethanol at’ -20°C for several hours, collected by centrifugation, and resuspended in 1 ml of 0.01 M-Tris (pH 7.6), 0.005 M-MgCl,, 0.1 “/b sodium dodecyl sulfate. The rRNA was next centrifuged through 5% to 20% sucrose gradients iii the same medium at 100,000 g for 10 h at 8°C in the SW27 rotor. The 18 S and 25 S rRNA fractions were collected separately by monitoring the gradient fractions at 260 nm, precipitamd in 2 vol. ethanol, and stored at - 20°C.
C!H,~RACTERIZ.~TTON
OF
(j) Ribosomal
THE
RLX;A/DNA
(‘OTTON
CENOME
*5ocl
h~ybridization
Ribosomal RNA labeled with lz51 was hybridized in 2 >: SSC to denatured DNA immobilized on Schleicher & Schuell BB, 24.mm filters (Gillespie & Spiegelman, 1965). The retention of DNA on the filters was greater than 907; as shown by duplicate experiments using labeled DNA. At the end of the hybridization reaction the filters were washed with 2 x SSC, incubated with 50 pg RNase A/ml (Sigma) for 30 min at 37°C in 2 x SS(‘, by the filters determinetl. washc,d again with 2 x SSC, air-dried, and the lz51 retained Routinely an extra blank filter (-DNA) was included on top of each group of react,iorl filizrs. since it, was lloted that 100 to 200 cts/min greater than hackgrourld were found on this top filter ( < 0.1 y. of the total input radioactivity). It is probable that some iodinatrri molecules were dried onto this top filter at 70°C during the reaction that could not I)(. subsequently removed. Ribosomal RNA/DNA hybridization kinetics were determined in solution in 1 x SS(!, 0.20,’o sodium dodecyl sulfate, at 60°C using denatured DNA that had been reduced to an averagcX length of 180 nucleotidea. The reaction mixtures were fract,ionated on hydroxylapatitc columns in 0.2% sodium dodecyl sulfate, and the extent of simultaneous DKA/ DNA reassociation determined as described in section (e), above. The radioactive rRNA ctluting in 0.12 x (unhybridized) and 0.48 M-PB huffcr (hybridized) was determined b,v scintillation comlting after precipitation of hydroxylapatit6~ rolilmn fractions \vit’h so,, t.richloroact:tic acid oIlto Milliporc filters.
3. Results (a)
DNA
conten.t~cotyledon
cell
In Figure 1 the growth of the cotton embryo, which is mostly comprised of its two cotyledons, is presented along with its cell number and DNA content per pair of cotyledons. Embryo growth is initially very slow hut, increases dramatically after day 20. The number of cells and DNA content per cotyledon pair also reflect this rapid increase in mass until about the 27t’h day. when cell division ceases. DNA content per cotyledon pair, however, continues to increase for about five more days Thus this increase in DNA before it reaches a plateau at about 9.4 pg DNBjcell. represents an increase in DNA per cell and. in Figure 1. appears to he a doubling prr cell. There are several lines of evidence tha,t this continued DKA synt’hwis in t,hts
Time after anthesis (days)
FIG. I. Parameters of cotton cotyledon development plot.ted UWMLSthe days after antheuis, a measure of embryo age. (------) Embryo weight); 0 ~~ :-I--, DNA/cotyledon pair; -- l -- l --, cell number/cotyledon pair.
510
\‘.
\VALHOT
ANI)
1,. R. I)URE
111
absence of cell division represents endoreduplication of the entire genome of cotton rather than the selective amplification of certain sequences. Firstly, the Cot, of nonrepetitive DNA isolated from cotyledon cells before and after the doubling of DNA content is identical. Secondly, no buoyant density satellites are detectable on analytical centrifugation analysis of .DNA isolated from cotyledon cells before and after the increase in DNA content per cell. Thirdly, the number of ribosomal RNA cistrons per unit of DNA is approximately the sa,mr in DNA isolated from these two developmental stages. The chromosomes of cells from mature cotyledons have not been examined to determine if the endoreduplication of the genome represents polytenization or polyploidization of the chromosomes, or both. Large increases in DNA per cell are not uncommon in the cotyledons of dicotyledonous embryos that consume their endosperm during embryogenesis. Furthermore, Millerd 6 Whitfeld (1973) have shown that this increase in Vi& faba cotyledons is also endoreduplication of the entire genome by the same criteria we have used. The establishment of the true ploidy level of DNA per cotyledon cell is not straightforward. First of all this species of cotton is tetraploid. Thus the DNA values per cell obtained from cotyledons undergoing cell division must represent some value between 4N and 8N. The final DNA per cell value of 9.4 pg per cell then would seem to reprosent some value between 8N and 16N. It is likely, however, t’hat the epidermal and parenchymal cells, the two main cell types of the cotyledonary tissue, reach different ploidy levels. The epidermal cells and their nuclei remain small during development, while parenchymal cells and their nuclei enlarge progressively (V. Walbot, unpublished data). Since the DNA content of plant nuclei is often correlated with nuclear and cell volume (Sparrow & Evans, 1961). epidermal cells probably remain at the 4N level. In addition, the vascular cells lose much of their contents, including nuclear DNA, during development. If it is assumed that only the parenchymal cells representing approximately two-thirds of the total, become polyploid, the average ploidy level of the parenchymal cells after endoreduplication would be about 16N. An alternative method for estimating the haploid genome size of cotton gives essentially the same result. Here, the reassociation kinetics of unique sequence cotton DNA are compared to the reassociation kinetics of a bacterial DNA of known genome size. At 350 nucleotide fragment length the Cot& (pure) of non-repetitive cotton DNA is 437 (Table 2): and the proportion of cotton DNA that is non-repetitive is 605:/b (Fig. 2). The C,f: of E. coli DNA is 4.2 by our measurements (Fig. 2), and the E. co& genome size is 2.8 x 10g (Cairns, 1963). Thus cotton non-repetitive sequence DNA is 104 times more complex than E. coli DNA (437/4.2) and the whole cotton genome contains 172 times more nucleotides than E. coEi (104/0.605). The minimum genome mass of cotton DNA (haploid DNA content) is, therefore, 4.8 x 1O1’ (172 x 2.8 x log), or 0,795 pg DNA/haploid genome. Using these figures, the average final ploidy level of all cotyledon cells taken together is about 12N (9.4 pg DNA per cell/O.795 pg per haploid genome). Using the same assumptions as above regarding the possible selective endoreduplication of parenchymal cells alone, the final ploidy of these cells would be 16iV. (b) Properties
of cotton DNA
DNA isolated from developing cotyledons, before and after endoreduplication, was analyzed by analytical CsCl density ultracentrifugation as was DNA from cotton roots.
CHARACTERIZATIOK
OF THE
(‘OTTOX
GENOME
511
E. coli K12 DNA gave a density of 1~708~0~001 g crnw3 under these conditions. Cotton DNA from all three sources gave essentially the same density of 1+693jO.O01 g cm-3. Further, the width of the DNA band from the three cotton sources appeared the same, indicating similar heterogeneity in each case and again suggesting that the increase in DNS content per cotyledon cell is a case of endoreduplication. The base composition of cotton DNA, calculated from the chromatographic separation of free bases produced by acid hydrolysis, has been reported to be 34qi (G i-C!). of which about one-fourth of bhe C (4.6:,,, of the total bases) is the methylated derivat’ive 5 MeC (Ergle & Katt,erman. 1961). Alt’hough bhe ba’se composition derived from our measurements of physical properties suggest a slightly higher G+C content. we have used the reported value of 4.6:/i, 5 MeC in correcting our measurements where possible. Unusual bases have been shown to influence the buo,vant density of DPL’X (Schildkraut et al., 1962). Kirk (1967) 1ias calculated that, methylation of 25q;, of the c,vtosine residues in a DNA of 50:/A (G rC) wi tl decrease t’hn buoyant density in CsCI of this DR;A by 4 mg cm -3. Szybalski & Yzybalski (197 1). comparing DXAs containing methylated bases, calculated that each lo,, met~hylation result’s in a decrease of 1 mg crne3 in buoyant density in CsCl. Another measurement of t’he magnit,ude of the effect of methylation has been made by comparing native chromosomal rDNA of Xenopus, which is 6776 (G+C). +5’?& 5 MeC. t(o the amplified rDXA which contains no 5 MeC (Dawid et al., 1970). The buoyant density of the chromosomal DIVA is 55 mg cmV3 less in neutral CsCI than t’he amplified rDNA. Based on the foregoing we have corrected the ohserved buoyant density by 5 mg cm-3 lo give l-698 g cm- :j. Prom this buoyant density. t,htl total G+C+5 MeC content- is 38.7”j, (Table 1) using Dhe equations of Schildkraut et ab. (1962). Th(l t, of native cotton DNA was found t,o be 85.6’C in 1 j’ SSC and 70~3°C in 0.1 ,y SSC. The drop in t, of 15.3 deg. C is close to the bheoretical value of 15.4 deg. C (Marmur & Doty, 1962). The dispersion. at’ cr 213: calculated by the method of Mahler & Dutton (1964), is 7.4 deg. C: indicating some het,erogeneit,y at the DNA molecular weight’ used (1 >, 107). Under similar conditions, native high molecular weight E. coli DRA gave a t, of 75~0°C in 0.1 >(: SSC, in good agreement with reported values, and gave a r~ 2/3 dispersion of 5.4 deg. C$ as expecbed for a simpler DNA. A calculation of the base composition of cotton DXL4 from the t, again requires that a correction for 5 MeC be made. The presence of 5 MeC increases the t, of the rDSA of Xenopus over t,he non-methylated rDNA 1)~ 1.5 deg. C in 0.1 x SSC (Dawid et nl.. 1970). Furthermore, synthetic polymers cont’aining 5 MeC show higher t, values than polymers lacking 5 MeC (Gill et al., 1974). When the t, of cotton DNA is , , for 4*6?4 5 MeC, the t, in 0.1 x SSC is lowered t’o 6&8”C, which indicates corrected a G-+C+5 MeC content of 36*lq/,. The base composition of cotton DNA has also been calculated from the equations of Felsenfeld (1971) applied t’o the native spectrum, which gives 36.4% (G+C), to the denatured spectrum which gives 34.6% (G+ C). and to the combination of native and denatured spectra which gives 38.576 (G-LC). The effect of 5 MeC on bhese analyses has not been systematically investigated. Table 1 shows that, t#he G+C+-5 MeC content calculated by all methods averages 36.906. (c) DNA/DNA
reassociation
Figure 2(a) shows the data obtained when the DNA/Dh’A reassociation of cot’ton DSX (averaging 350 base-pairs) is measurrd 1)~ hydroxylapat,it,e chromatography
51.5
36.1
Huoyant
51.0
38.7
tlensityf
of (l- 1-C+5
60.8
36.4
Native
MeC
34.6
DNA
X3.5
$ Buoyant
t DXA
(1962).
Spectral andysia# Native and denat~ured
in cotton
1)enaturetl
content
sitmplrs in 0.1 .I SS(‘; t, of cotton l>XA corrcctncl for 5 MeC’. C’alculated from equation of Marmur & Doty dtwsity of cotton l>XL4 corrected for 5 Md’. C’nlculatwl from t,he cqmt~ion of Schildkraut et d. (1961). 9 Based on the equations of Feluenfeld (1971). * Equal weight givrn to each method of rstimatrrl 0 it c’ + 5 &Id’.
IC. coli I
Biol.
(1976)
101, 503-536
Developmental Biochemistry of Cotton Seed Embryogenesis and Germination VI1.t Characterization
of the Cotton Genome
VIRGINIA WALBOT~ AND L. S. UURE III$ I)epartment
of Biochemistry.
(Received 30 April
Univer&y
nf (r’eorqia, Athew,
(!a 30602, [J.S.A.
197.5, and in revised form 20 September 1975)
The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, baxe composition, and genetic complexity. The haploid genome size is found to be 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due t,o trrrdored~lplicatic)n of the entire penomo. Non-repetitive deoxynucleotide sequences account for approximately 60.5qO of the cotton genome (C& pure 7 = 437); highly repetitive sequences ( > 10,000 r(bpetition frequency) constitute about 7.7 Y. of tile penomc (C& pure = 4.6X IO 4, and intermediately repetitive sequences constitute tile remaining 27 “/b of cytoplasmic ribottle genome (C,t+ pure = 1.46). Hybridizat,ion of ‘251-labrletl somal RNA to whole-cell DNA on filters and in solution indiratr approximately 300 to 350 copies of the rRNA cistrons per haploid genome. The interspersion of repetitive sequences that reassociate between C,t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined t)y determining t,he reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the penomr consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 200/b of the genome may bc involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence di\.cLrpence is evident, among t,he members of this component. Highly repetitive sequence elements that are reassociated by a C,t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of tllis highly repetitive component.
11Abbreviations used: C,t, product of DNA concentration (mol nucleotido I ‘) x time(s); C,t,, that C,t value at which one-half of a kinetic component of DNA will have reassociated: Coti pure, that C,t value at which one-half of a kinetic component of DNA would have re: associated if only thet component were present, calculated as the product of Cot l x the fraction of t’he genome in that kinetic component; 5 MeC, 5.met,hylcytosine; rDNA, ribosomal RNA cist,rons. 503
1. Introduction All the genomes of euknryotes thus far examined contain both repetit#ive and nonrepetitive sequence elements. The non-repetitive sequence elements are thought to include t’he structural genes. There is some evidence for t,his hypothesis, since the messenger RNAs coding for specific proteins are transcribed from non-repetitive DNA (globin, Bishop & Freeman, 1973: Bishop & Rosbash. 1973; Harrison af al.. 1974: ovalbumin, Harris ef al., 1973: fibroin. Suzuki et aZ., 1972). Furthermore, mRNA isolated from various tissues has been shown t,o be transcribed primarily from non-repetitive DNA sequences, although some repetitive copy transcript is also present (Greenberg & Perry, 1971; Goldberg et al., 1973; Firtel & Lodish, 1973: Bishop et al., 1974: Campo & Bishop. 1974; Galau et al., 1974: Klein et al.; 1974: Spradling et al., 1974; Davidson et al.. 1975). Messenger RNA for histone proteins has been shown to be transcribed from repetitive copy DNA (Weinberg et al., 1972). The proportion of transcripts of repetitive DNA elements in heterogeneous nuclear RNA is higher than from mRNA from t’he same tissue (,Jelinek et ~2.. 1973; Holmes & Bonner, 1974; Smith et al., 1974) suggesting that initial transcriptional products contain more RNA transcribed from repetitive DNA than does functional mRNA. The two kinetic components of the eukaryoOic genome are interspersed to some extent in all the animal genomes which have been analyzed in d&ail. Interspersion has been demonstrated (1) by the increasing proportion of DNA binding to hydroxylapatite at low C,t values? with DNA fragments of increasing length (calf, Britten & Smith, 1970: Xenopus, Davidson et al., 1973: sea urchin, Graham et al.. 1974: Oncopelfus fusciatus, Lagowski et al., 1973: chicken, de *Jimenez et al.: 1974), (2) by electron microscopy of reassociated DNA (rat liver, Bonner sf al., 1973: DrosophiZa, Manning et al.? 1975) and (3) by inference from the presence of both repetitive and non-repet’it,ive sequences transcribed into the same RNA molecules (Dictyostelium, Firtel & Lodish, 1973). In both Xenopus (Davidson et al.: 1973) and sea urchin (Graham et al.. 1974) short repetitive sequences of about 300 base-pairs are interspersed with longer non-repetitive sequences of about 1000 base-pairs in over 5Oy{, of the genome. Regions of clustered repetitive sequences, a longer interspersion pattern (repetitive DNA elements more than 4000 bases apart) and long sttret8ches of non-repet’itive DNA also probably exist. .It is attractive to view t’he interspersion pattern of repetitive a,nd non-repetitive sequence elements as a contiguous arrangement of regulatory (repetitive) and structural gene (non-repet,itive) sequences (reviewed by Davidson & Britten, 1973). This notion is strengthened by the fact that most mRNA sequences in SW urchin gastrulae are transcribed from non-repctitivct DNA adjaccant, to repetitIivcb DNA (Davidson et wZ., 1975). t Abbreviations used: C,t, product of Di’GA concentration (mol nucleotlde 1 -‘) x time(s); C,t*, that Cot value at which one-half of a kinetic component of DNA will have reassociated; Cat+ pure, that Got value at which one-half of a kinetic component of DNB would have reassociated if only that component were present,, calculated as the product, of C,l& x the fraction of the genome in that kinetic component: 5 MeC, B-methylcytosine; rDNA, ribosomal RNA oistrons.
(‘HARBC’TERIZSTIOK
OY THE
GROTTOS
GENOME:
50.5
Although the presence of repetitive and non-repetitive DI\;A has also been report.cd in higher plants (Britten & Kohne, 1967; Sivolap & Bonner, 1971; Millerd & Whitfeld. 1973; Flavell et al., 1974; Cullis & Schweizer, 1974; Nze-Ekekang et al., 1974; Ranjekar ut al.. 1974) bht: possible interspersion of t,hese components has not been studied. Tn this paper we present evidence demonstrating the ext,ensive int’erspersion of thcb mildly,, repctit,irfB and non-rep&t ive sequcnc(’ elements of t,he cot’ton penomr:.
2. Materials and Methods (a) Plant material (:rt,c~rlliouse-g~,(~~~~ plants of ~hssypium I&SU~WL ((‘oker var. %Ol) provided a contillnous suppl?- of developing embryos md mature seed. The growt’il rate of cott,on embryos insidtx the cott,on boll is shown in Fig. 1 ; sevc~ral hiochemion1 paralncxtcrs of elnbryo dc\-elopmont Iravc IHWI studied (Dure. 1973). (I)) Cell num6er
tleter),li)lntiorrs
Cotyledons from embryos dissected at various stages of development were digest,rd for 48 h in 596 cllromic acid to disrupt the t,issue t,o sirlgle cells (Brown & Rickless, 1949). The cotyledons consist of epidermal, parenchymal and vascular cells. Dilutions of the ccl1 suspensions were counted in a Fuchs-~Rojenthal hemacytometer at a magnification of 100. From 5 to 10 dilutions were made of each cotyledon cell suspension and 5 port’ions of eaclr dilution were countsed. Approximately 2:; of the cells are vascular cells, xylem oleme,lts and sieve tubes, t-hat do not contain an)- cell orga,nelles or nucleic acids; thesr cells \\.erc tlot incaludcd in the total cell number. since tllsy do not) contribute to total DNA c!clut~nlll.
(c) D,VA
extraction.
purijcation
and~rag,nentation
DNA was prepared from embryonic cotyledons at various stages of development. from cotyledons of the mature seed. and from germinating root tips. Homogenizat’ion was carried out in 1 x SSC (SSC is 0.15 M-NaCl, 0.015 Al-trisodium citrate), 2.57; sodium dodecyl sulfate, 1 mM-EDTA (25 g fresh wt/250 ml) with a Polytron homogenizer (Brinkman Instruments) at top speed for 2 min. The homogenate was stirred at room temperaturtj for several hours. then made 1 31 in NaC104 and heated to 60°C for 15 min. Two vol. chloroform/inoamyl alcohol (24:l) were added to the Ilomogenate and the mixture stirred for sex-era1 hours. The aqueous phase was recovered b>- centrifugation at 5000 g for 10 milt and re-extracted wit,h chloroform/isoamyl alcohol for I h. The aqueous phase was again recovered as described above and the nucleic acids precipitated by the addition of 2.5 vol. cold ethenol. The precipitate was resuspended in 20 ml of 1 >; SW and incubated with 2 mg pancreatic RNcse (Sigma), boiled for IO min prior to use. and 400 units T, RNastb (Wort~llingt~on) for 2 11 at 37”(.‘. Aft)er several additional chloroform/isoamyl alcohol extra&ions and ethanol precipitations, from which tllr: DNA was collected by spooling, t,he DNA was purified from residual oligoribonucleotides by pmcipitat,ion with 0.54 vol. isopropanol (Marmur, 1961). Finally the DNA was banded in preparative (IsCl gradients h5, centrifugation at 75,000 g for 60 h at 22°C. Gradient fractions containing DNA were pooled. dialyzed extensively against 1 x SSC, and stored as &anol precipitates. The, weight average molecular weight of native DNA samples was determined b!, sedimentation analysis with a Spinco model E analytical ultracent~rifuge equipped wit11 an ultra.violet light scanner, using the equations given by Studier (1965). Single-stranded weight average molecular weight was determined in O.!) .w-NaCI, 0.1 &I-NaOH, and doublest,randed molecular weight in 1-O M-NaCl. Three determinations were made for each sample. The weight average molecular weight of native DNA isolated b>- our procedure nvcragtxd 1 > 107. Singl+stranded molecular wcaiphts averaged al)ont 40 to X0, of tllr nativcl DNA \‘HIll(~S. :i I
306
\‘.
\\‘;\l,BOT
AXI)
L.
8. DURE
III
DNA blloyatlt tlensit,y \WS tlrtrrmined by isopycnic C&l centrifugation by the method of Szybalski & Szybalski ( 1971) using a Spinco model E ultracentrifuge equipped with a DNA was used as a marker (1.731 g cmW3); data reported u.\-. scanner. ~~icrococcus l&us are the average of 3 or more crntrifugations on 2 or more separately prepared DNA samples. The optical charact,eristics of cotton DNA were obtained with a Gary 15 spectrophotoInetcr. The percentage hyprrchromicity and melting temperature (t,) were determined by melting DNA samples in a water-,jackcttcd cuvette attached to a Lauda K2jR circnlnting water bath. A thermistor probe (YSI model 402) was inserted into the solution tin-ough a Teflon plug at, tllc? top of thr cuvet~te. The probta was connected to a telethermonlrtrr (YSl model 4286) and temperature readings recorded manually. The dispersiotl, (T2,3r was determined as described 1)~ Mahlrr & D~ltton (1964). Escherichin coli DNA bvas optically analyzed as a standard. Native DNA was sheared to various sizes by sonic&ion, by passage tllrough a 25 gauge syringa needle, or by higll-speed blending in a Virtis llomogenizer as described by Britten et a/. (1974). Ttrr single-stranded lrr@lr of t,he DNA fragments was dct~erminrd in isoet ul.. 197X) 01' kinetic SIIC~OSO gradients corltainillg 0.1 Izr-NaOH (Noll, 1967 ; Davidson I)y analyt,ical ~iltrac~rltrif~l~at,ioll. The equations relating molt~cutar wtaight to srdimerltation rate (Studier, 1965) \vcArcaderived for molecules more than an order of mapnitllde larger than the smallest molecules used in t’his st,udy ; thus. the measurements reported reflect relative size relationships, rather t,han ttle absolute molecular size. Fragments produced by all 3 methods of shearing showed thermal denaturatioll charactorist,ics similar to nativrl cotton DNA (Fig. 3(a)), indicating t)bat the> fragmentas were ttssontiatly undenatured. (d) lo&nation
of nucleic
ucirl~s
Heat,-denatured DNA alld purified rRNA were iodinated by tlje procedure of C&z et ul. lzsI ( 17.7 Ci/mmol, New England Nuclear) itI ( l!j72) wit11 the following modifications. NaOH was neut,ralized wit11 0.1 M-HCl in 0.005 M-Na,SO, to reduce the-ioditie to bypoiodate, the reactive oxidation stat,e for t,he iodination reaction; the thallimn chloride (K & K Laboratories) concentration was reduced to 10m4 ~1. Following the reaction, the iodinatc>d nucleic acids were separated from other reaction components by chromatography on Hephadex G25 in 0.1 x SSC and their molecular weight determined again. Short fragment length DNA ( < 1600 nucleotidos) showed no detectable reduction in size during iodination ; however, longer fragment length DNA did (30% reduction at initial length of 9200 nuclootides). The ribosomal RNAs showed some decrease in molecular weight after iodination, as shown hy their rnigratioll on polyacrylamide gels. DNA iodinatod in this way did not exhibit any change in specific activity ( + 2yo) after boiling for 10 min in a sealed capillary or prolonged incubation at 60°C in 0.12 >l-PB buffer (equimolar amount,s of mono- and dibasic phosphate buffer, pH 6.8). Cotton DNA is approximately 130/b cytidine and 4.6% 5.methylcytidine (see Results). Assuming that the 5-MeC residues are not iodinated, a spec. act. of 5 x lo5 cts/min per pg DNA is equivalent to one [1251]iodocytidine and 21 iodocytidine residues for every 3800 cytidine residues. Iodination is assumed to be basically a random process, at least with respect, to the labeling of the major kinetic components of cotton DNA. This was confirmed by the fact that the specific activities of mildly repetitive sequence DNA (reassociating between C,t values of 0.1 and 50) and non-repetitive sequence DNA (not reassociated by Cot z 50) were identical. However, since the iodination react’ion entails an incubation that equates to a C,t value of 0.05, the most rapidly reassociating DNA component (zero-time: binding which, in turn, could lead to an DNA) may not participate in the iodination reaction, underestimation of the magnitude of this component when measured isotopically.
(e) DNA/DNA
reaesociation
The rate of reassociation of heat-denatured DNA fragmented to several size classes was determined. Portions of sheared DNA were sealed in sterile capillary tubes or in ampules, denatured in a boiling water bath for 7 min, and then allowed to renature for
(‘HARACTERIZATION
OF
THE
COTTON
GENOME
507
varying lengths of time. The reassociation reactions were terminated by freezing the samples at - 20°C. The criterion conditions (Britten & Kohne, 1967) for these experiment,s were 0.12 M-PB buffer, 60°C (t,-225 deg. C). Various concentrations of SSC and PB buffer were used in the renaturation experiments and all values reported have been corrected to the Cot value equivalent to criterion conditions. In addition, the apparent rate of reassociation has been corrected to accolmt for the low G $ (’ content of cott,on DNA using the formula of Wetmur & Davidson (1968). Reassociated molecules were separated from single-stranded molecules on hydroxylapatite (BioRad) columns prepared in 0.12 M-PB buffer and 0.1% sodium dodecyl sulfat)t! at 60°C’. The hydroxylapatite was boiled for 5 min prior to pouring into the column and the column was washed with 0.12 M-PB buffer to remove sodium dodecyl sulfate prior to loading the sample. Single-stranded molecules were eluted with 0.12 M-PB and thcl reassociated duplexes &ted with 0.48 M-PB (Britt’en et al., 1974). The DNA content of tlach fraction was determined spectrally and/or by the determination of lz51 cont,ent. Recovery of radioactivity and absorbancy units was 1OO& lOq/,. In experiments in which lz61-labeled DNA was used as tracer, it was less than O.OSqh the concentration of thaw unlabeled fragments. DNA of fragment length f-PB buffer and then the fraction elutinp in 0.48 M-PB buffer (2) collected and its radioactivity determined. In subsequent experiments, to rstimate the binding due to reassociation exclusive of Z, its contribution to the observed reassociation was stibtracted as described by Davidson et al. (1973). The proportion of DNA retained by the hydroxylapatite colllmn due to %: varies with fragment length (Fig. 4), and for experiments with larger fragment lengths (> 1500 nucleotides) zero-time binding DNA was removed from the lZ51-labeled preparations by recovering and losing the fraction not bound to the hydroxylapatite column at Cot < 10m5. These t This program was the “General Multiparametric Curve-Fitting Program CFTB” developed by L. M&es and published by the Computing Laboratory, Department, of Chemistry. Clarkson College of Technology, Potsdam, N.T. 13676, U.S.A.
5OS “&ripped”
1.. \V.AJ,HO’l preparations for wliich
((I.1 t,o W25yA)
(g)
.\SIl
1,. R. 1)UHE
contained a small \&able fraction no corrnct,iori Ilas t,etsri ma&~.
Characterization
oj” reasmciated
DNA
IL1 of zero-t*ime
hy S, m&ease
binding
material
digestion
DNA samples (0.5 to 1 mg DNA) reassociated to a Cot value of 0.1, 10 or 50 in 0.3 btNaCl, 0.01 M-PIPES buffer (Sigma), pH 6.7 (Davidson et al., 1973) were incubated uitli 200,000 units of S1 nuclcase (Miles Laboratories) in a solution containing 0.2 iw-NaCl, 0.0167 ~I-PIPES buffer, 0.067 mai-zinc acetate, aiid 1.85 mM-mercaptoethanol for 3 Ii at. 37°C’ in a scaled ampule. Tlrc digested samplos were loaded directly on a Sephadex G75 column equilibrated in 0.12 N- PB buffer. The void voliuno contained 8, nuclease-resistant duplex DNA with few, if any, single-stranded regions as determined by electron microscopy. The proportion of Xi nuclease-resistant to S,-sensititre material was determined by a comparison of the absorbance at 260 nm of the fractions containing duplex DNA to the totally included fraction whicli contained all the digestion products. In some experiments 1251-labeled DNA samples were used and the separation and the relative amounts of material contained in t’he 2 fract)ions determined hp monitoring radioactivit>y. (11) Electron
w~icroscopy
was performed as described 1,s Visualization of DNA samples by electron microscopy Davis et al. (1971). Aqueous samples containing 0.5 pg duplex DNA/ml, 0.5 M-ammonium acet,ate, 0.001 M-EDTA(Na,), 6 mg cyt~ochromo c/ml (Calbiochem), pH adjusted to 7.6, wt~~: spread on a 0.25 M-ammonium acet,ate Iiypophase. Samples containing both single and double-stranded DNA were prepared in a solution containing 0.5 pg DNA/ml, 0.1 M-Tris, 0.01 M-EDTA(Na,), 6 mg cytochrome c/ml and 32.5 46 formamide (pH 8.5). This COIICCILtration of formamide at room temperature mimics t,ticl criterion temperature for hydroxylapatite chromatography, since cacti percent by volume of formamidu reduces the t, by 0.72 deg. C (McConaughy et al., 1969). At the ionic strength of the samples, the t, of cotton DNA would be about 70°C. Ttlc: 3~2.5~4 formamido would reduce this to ahod 25 deg. C’. Thus mismatched duplexes 47”C, making room temperature roughly equal t,o t, kvould be maintained to t,ho same extent as under criterion conditions. These samples were spread over a hypopha,se containing 0.01 31.Tris, 0.001 nr-sodium acetate and Ifi”/; acotato and rotary formamide (pH 8.5). All samples wer(’ staiued wit,h 10V6 w-many1 shadowed with PtjPd (4: 1) prior to rxaminat,ion in a Philips 200 electron microscope. Lengths of cotton DNA molecules were calculated from calibrated diffraction gratings, and by comparison to +X 174 single-stranded, assuming 2.07 x 106 daltons of DNA/pin. closed circiilar DNA. 1.7 x 1O6 dalmns. or co1 El plasmid, dont)l~?-strRlldod closed circular DNA, 4.2 x IO6 daltorw. Tlic> two mrtliotls of cst,imating DNA lengtti agreed lvittiirr 5’j,,. (i) lZiboaoma1 RNA
%solation
Monomeric 80 S ribosomes were isolated from 3 g of cotyledons by liomogenizatiou in 24 ml of 0.1 nr-Tris (pH 8.0), 0.005 1~-M&l,, 0.01 M-KC1 and 0.5% Triton X-100 (Rohm & Haas), followed by layering 4-ml portions of t,ht: liomogenate on 3(1-d linear sucrose gradients (10% to 40% sucrose containiug 0.01 nr-Tris (pH 8.0), 0.005 %I-MgCl,, 0.01 M-KC]) and centrifugation for 6 h at 100,000 g in the Beckman SW27 rotor. Fractionation of the gradients was monitored at 260 nm and fractions containing 80 S ribosomes were pooled and the ribosomes pelleted by centrifugation at 250,000 g for 12 h at 4”C, in the Beckman Spinco 60Ti rotor. The ribosomal pellets were dispersed in 0.01 ivr-Tris (pH 7.6), 0.005 MMgCl,, 0.1% sodium dodecyl sulfate and extracted with buffer-saturated phenol for 30 min at 4°C. The aqueous phase was recovered by oentrifugation at 10,000 g for 10 min and the extraction repeated. The rRNA was precipitated with 2 vol. ethanol at’ -20°C for several hours, collected by centrifugation, and resuspended in 1 ml of 0.01 M-Tris (pH 7.6), 0.005 M-MgCl,, 0.1 “/b sodium dodecyl sulfate. The rRNA was next centrifuged through 5% to 20% sucrose gradients iii the same medium at 100,000 g for 10 h at 8°C in the SW27 rotor. The 18 S and 25 S rRNA fractions were collected separately by monitoring the gradient fractions at 260 nm, precipitamd in 2 vol. ethanol, and stored at - 20°C.
C!H,~RACTERIZ.~TTON
OF
(j) Ribosomal
THE
RLX;A/DNA
(‘OTTON
CENOME
*5ocl
h~ybridization
Ribosomal RNA labeled with lz51 was hybridized in 2 >: SSC to denatured DNA immobilized on Schleicher & Schuell BB, 24.mm filters (Gillespie & Spiegelman, 1965). The retention of DNA on the filters was greater than 907; as shown by duplicate experiments using labeled DNA. At the end of the hybridization reaction the filters were washed with 2 x SSC, incubated with 50 pg RNase A/ml (Sigma) for 30 min at 37°C in 2 x SS(‘, by the filters determinetl. washc,d again with 2 x SSC, air-dried, and the lz51 retained Routinely an extra blank filter (-DNA) was included on top of each group of react,iorl filizrs. since it, was lloted that 100 to 200 cts/min greater than hackgrourld were found on this top filter ( < 0.1 y. of the total input radioactivity). It is probable that some iodinatrri molecules were dried onto this top filter at 70°C during the reaction that could not I)(. subsequently removed. Ribosomal RNA/DNA hybridization kinetics were determined in solution in 1 x SS(!, 0.20,’o sodium dodecyl sulfate, at 60°C using denatured DNA that had been reduced to an averagcX length of 180 nucleotidea. The reaction mixtures were fract,ionated on hydroxylapatitc columns in 0.2% sodium dodecyl sulfate, and the extent of simultaneous DKA/ DNA reassociation determined as described in section (e), above. The radioactive rRNA ctluting in 0.12 x (unhybridized) and 0.48 M-PB huffcr (hybridized) was determined b,v scintillation comlting after precipitation of hydroxylapatit6~ rolilmn fractions \vit’h so,, t.richloroact:tic acid oIlto Milliporc filters.
3. Results (a)
DNA
conten.t~cotyledon
cell
In Figure 1 the growth of the cotton embryo, which is mostly comprised of its two cotyledons, is presented along with its cell number and DNA content per pair of cotyledons. Embryo growth is initially very slow hut, increases dramatically after day 20. The number of cells and DNA content per cotyledon pair also reflect this rapid increase in mass until about the 27t’h day. when cell division ceases. DNA content per cotyledon pair, however, continues to increase for about five more days Thus this increase in DNA before it reaches a plateau at about 9.4 pg DNBjcell. represents an increase in DNA per cell and. in Figure 1. appears to he a doubling prr cell. There are several lines of evidence tha,t this continued DKA synt’hwis in t,hts
Time after anthesis (days)
FIG. I. Parameters of cotton cotyledon development plot.ted UWMLSthe days after antheuis, a measure of embryo age. (------) Embryo weight); 0 ~~ :-I--, DNA/cotyledon pair; -- l -- l --, cell number/cotyledon pair.
510
\‘.
\VALHOT
ANI)
1,. R. I)URE
111
absence of cell division represents endoreduplication of the entire genome of cotton rather than the selective amplification of certain sequences. Firstly, the Cot, of nonrepetitive DNA isolated from cotyledon cells before and after the doubling of DNA content is identical. Secondly, no buoyant density satellites are detectable on analytical centrifugation analysis of .DNA isolated from cotyledon cells before and after the increase in DNA content per cell. Thirdly, the number of ribosomal RNA cistrons per unit of DNA is approximately the sa,mr in DNA isolated from these two developmental stages. The chromosomes of cells from mature cotyledons have not been examined to determine if the endoreduplication of the genome represents polytenization or polyploidization of the chromosomes, or both. Large increases in DNA per cell are not uncommon in the cotyledons of dicotyledonous embryos that consume their endosperm during embryogenesis. Furthermore, Millerd 6 Whitfeld (1973) have shown that this increase in Vi& faba cotyledons is also endoreduplication of the entire genome by the same criteria we have used. The establishment of the true ploidy level of DNA per cotyledon cell is not straightforward. First of all this species of cotton is tetraploid. Thus the DNA values per cell obtained from cotyledons undergoing cell division must represent some value between 4N and 8N. The final DNA per cell value of 9.4 pg per cell then would seem to reprosent some value between 8N and 16N. It is likely, however, t’hat the epidermal and parenchymal cells, the two main cell types of the cotyledonary tissue, reach different ploidy levels. The epidermal cells and their nuclei remain small during development, while parenchymal cells and their nuclei enlarge progressively (V. Walbot, unpublished data). Since the DNA content of plant nuclei is often correlated with nuclear and cell volume (Sparrow & Evans, 1961). epidermal cells probably remain at the 4N level. In addition, the vascular cells lose much of their contents, including nuclear DNA, during development. If it is assumed that only the parenchymal cells representing approximately two-thirds of the total, become polyploid, the average ploidy level of the parenchymal cells after endoreduplication would be about 16N. An alternative method for estimating the haploid genome size of cotton gives essentially the same result. Here, the reassociation kinetics of unique sequence cotton DNA are compared to the reassociation kinetics of a bacterial DNA of known genome size. At 350 nucleotide fragment length the Cot& (pure) of non-repetitive cotton DNA is 437 (Table 2): and the proportion of cotton DNA that is non-repetitive is 605:/b (Fig. 2). The C,f: of E. coli DNA is 4.2 by our measurements (Fig. 2), and the E. co& genome size is 2.8 x 10g (Cairns, 1963). Thus cotton non-repetitive sequence DNA is 104 times more complex than E. coli DNA (437/4.2) and the whole cotton genome contains 172 times more nucleotides than E. coEi (104/0.605). The minimum genome mass of cotton DNA (haploid DNA content) is, therefore, 4.8 x 1O1’ (172 x 2.8 x log), or 0,795 pg DNA/haploid genome. Using these figures, the average final ploidy level of all cotyledon cells taken together is about 12N (9.4 pg DNA per cell/O.795 pg per haploid genome). Using the same assumptions as above regarding the possible selective endoreduplication of parenchymal cells alone, the final ploidy of these cells would be 16iV. (b) Properties
of cotton DNA
DNA isolated from developing cotyledons, before and after endoreduplication, was analyzed by analytical CsCl density ultracentrifugation as was DNA from cotton roots.
CHARACTERIZATIOK
OF THE
(‘OTTOX
GENOME
511
E. coli K12 DNA gave a density of 1~708~0~001 g crnw3 under these conditions. Cotton DNA from all three sources gave essentially the same density of 1+693jO.O01 g cm-3. Further, the width of the DNA band from the three cotton sources appeared the same, indicating similar heterogeneity in each case and again suggesting that the increase in DNS content per cotyledon cell is a case of endoreduplication. The base composition of cotton DNA, calculated from the chromatographic separation of free bases produced by acid hydrolysis, has been reported to be 34qi (G i-C!). of which about one-fourth of bhe C (4.6:,,, of the total bases) is the methylated derivat’ive 5 MeC (Ergle & Katt,erman. 1961). Alt’hough bhe ba’se composition derived from our measurements of physical properties suggest a slightly higher G+C content. we have used the reported value of 4.6:/i, 5 MeC in correcting our measurements where possible. Unusual bases have been shown to influence the buo,vant density of DPL’X (Schildkraut et al., 1962). Kirk (1967) 1ias calculated that, methylation of 25q;, of the c,vtosine residues in a DNA of 50:/A (G rC) wi tl decrease t’hn buoyant density in CsCI of this DR;A by 4 mg cm -3. Szybalski & Yzybalski (197 1). comparing DXAs containing methylated bases, calculated that each lo,, met~hylation result’s in a decrease of 1 mg crne3 in buoyant density in CsCl. Another measurement of t’he magnit,ude of the effect of methylation has been made by comparing native chromosomal rDNA of Xenopus, which is 6776 (G+C). +5’?& 5 MeC. t(o the amplified rDXA which contains no 5 MeC (Dawid et al., 1970). The buoyant density of the chromosomal DIVA is 55 mg cmV3 less in neutral CsCI than t’he amplified rDNA. Based on the foregoing we have corrected the ohserved buoyant density by 5 mg cm-3 lo give l-698 g cm- :j. Prom this buoyant density. t,htl total G+C+5 MeC content- is 38.7”j, (Table 1) using Dhe equations of Schildkraut et ab. (1962). Th(l t, of native cotton DNA was found t,o be 85.6’C in 1 j’ SSC and 70~3°C in 0.1 ,y SSC. The drop in t, of 15.3 deg. C is close to the bheoretical value of 15.4 deg. C (Marmur & Doty, 1962). The dispersion. at’ cr 213: calculated by the method of Mahler & Dutton (1964), is 7.4 deg. C: indicating some het,erogeneit,y at the DNA molecular weight’ used (1 >, 107). Under similar conditions, native high molecular weight E. coli DRA gave a t, of 75~0°C in 0.1 >(: SSC, in good agreement with reported values, and gave a r~ 2/3 dispersion of 5.4 deg. C$ as expecbed for a simpler DNA. A calculation of the base composition of cotton DXL4 from the t, again requires that a correction for 5 MeC be made. The presence of 5 MeC increases the t, of the rDSA of Xenopus over t,he non-methylated rDNA 1)~ 1.5 deg. C in 0.1 x SSC (Dawid et nl.. 1970). Furthermore, synthetic polymers cont’aining 5 MeC show higher t, values than polymers lacking 5 MeC (Gill et al., 1974). When the t, of cotton DNA is , , for 4*6?4 5 MeC, the t, in 0.1 x SSC is lowered t’o 6&8”C, which indicates corrected a G-+C+5 MeC content of 36*lq/,. The base composition of cotton DNA has also been calculated from the equations of Felsenfeld (1971) applied t’o the native spectrum, which gives 36.4% (G+C), to the denatured spectrum which gives 34.6% (G+ C). and to the combination of native and denatured spectra which gives 38.576 (G-LC). The effect of 5 MeC on bhese analyses has not been systematically investigated. Table 1 shows that, t#he G+C+-5 MeC content calculated by all methods averages 36.906. (c) DNA/DNA
reassociation
Figure 2(a) shows the data obtained when the DNA/Dh’A reassociation of cot’ton DSX (averaging 350 base-pairs) is measurrd 1)~ hydroxylapat,it,e chromatography
51.5
36.1
Huoyant
51.0
38.7
tlensityf
of (l- 1-C+5
60.8
36.4
Native
MeC
34.6
DNA
X3.5
$ Buoyant
t DXA
(1962).
Spectral andysia# Native and denat~ured
in cotton
1)enaturetl
content
sitmplrs in 0.1 .I SS(‘; t, of cotton l>XA corrcctncl for 5 MeC’. C’alculated from equation of Marmur & Doty dtwsity of cotton l>XL4 corrected for 5 Md’. C’nlculatwl from t,he cqmt~ion of Schildkraut et d. (1961). 9 Based on the equations of Feluenfeld (1971). * Equal weight givrn to each method of rstimatrrl 0 it c’ + 5 &Id’.
IC. coli I
